Understanding the pathophysiology and developmental biology of the vascular system is a major focus of biomedical research. In addition to cardiovascular disease, vascular pathology is a hallmark characteristic of cancer development, progression and metastasis. Tumors must develop and maintain new blood vasculature in order to meet basic nutrient and oxygen demands and to eliminate toxic byproducts of rapid metabolism. Blood vessel growth is influenced by factors secreted in the tumor, stroma and endothelial cells. Tumors also metastasize to other regions of the body through the blood vasculature, and disseminated disease is the most common cause of death due to cancer. Studies have shown correlations between vessel density and poor prognosis in some, but not all cancers. There is significant focus on different methods for characterization of blood vessels by in vitro methods (discussed further below) and blood flow by in vivo imaging methods such as contrast-enhanced ultrasound imaging or dynamic contrast enhanced (DCE)-MRI as it is essential for understanding of tumor progression and developing new diagnostic and therapeutic approaches.
Angiogenesis is the development of new vasculature from existing vasculature. In this process, endothelial cells sprout from preexisting vessels, proliferate and form new vessels. In addition to being an essential function of normal developmental physiology, angiogenesis is of central importance to the growth of tumors, as failure to develop new vasculature limits growth of solid tumors to less than 2-3 mm. Angiogenic vessels have irregular structures with abnormal blood flow and heterogeneous distribution. They tend to be easily compressed, resulting in hypoxia and heterogeneity in blood flow. In recognition of this, drugs intended to inhibit angiogenesis have been developed and are used to treat various human cancers. This therapeutic avenue remains an important component of drug discovery and development and several anti-angiogenesis drugs are now FDA approved for certain cancers (e.g. Bevacizumab (Avastin or Ramucirumab (Cyramza). While much progress in the translation of angiogenesis research to clinical applications has been made, available treatments targeting angiogenesis in cancer achieve relatively modest efficacy in the clinic.
Typical vasculature characterization methods involve immunohistochemical staining of protein antigens normally expressed by the endothelial cells of the blood vessel wall. Common antigens used to characterize vessels include CD31 (PECAM1), CD34 (Hematopoietic progenitor cell antigen CD34), and von Willebrand Factor (vWF—Factor VIII Related Antigen). The characterization and quantification of vascular features using this protein expression information includes micro vessel counts, microvessel density (MVD) and morphology metrics (size, dimension etc). Correlated protein expression (e.g. VEGF) patterns is also an important aspect of understanding effects of angiogenesis on local biology. Quantitation methods including direct immunohistochemistry-based microvessel density, have been used, whereby microvessels/endothelial cells are counted in a standardized grid using light microscopy and expressed as microvessel density (MVD). Microvessel density (MVD) has been found to be associated with poorer prognosis in some but not all cancers. To date, MVD has not been shown to be a valid measure to guide or evaluate anti-angiogenic treatment; the complex geometry and heterogeneity of tumor vasculature mean that vascular network cannot be measured by MVD counts alone.
Further, while the previously described proteins are expressed by endothelial cells, each is limited by certain shortcomings. For example, CD31 is considered to be universally expressed by all endothelial cells of the blood vasculature. However, it is also expressed by several cells of the immune system (e.g., monocytes, granulocytes, lymphocytes). This limits its utility in automated image analysis algorithms, as cells other than those comprising vessels can be erroneously classified as endothelial cells. In addition, in fluorescence microscopy, anti CD31 antibodies often stains discontinuous segments of cells, including incomplete lining of the vessel lumen and endothelial cell junctions. It is therefore challenging to segment individual vessels stained by anti-CD31 immunofluorescence; segmentation results in fragments of vessels, resulting in overestimation of vessel number and underestimation of vessel size. Similar problems are found in immunofluorescence targeting CD34, a marker of blood vessel progenitor cells, hematopoietic stem cells, white blood cells and certain fibroblasts. von Willebrand Factor staining characteristics pose an additional challenge: endothelial cells of immature neoangiogeneic vasculature do not express vWF, resulting in underestimates of vessel counts if used for image segmentation and vessel quantification. Stains of the additional proteins listed above are all limited by similar problems, and other than staining CD31, CD34, and vwF on separate tissue sections and correlating MVD results, no universal marker has been advanced to solve the issues described. These problems are universally encountered by scientists and clinicians seeking automated approaches to sole vessel analysis problems, limiting progress in vessel biology research.
Therefore, cancer biologists still seek to better understand the cancer angiogenesis, inhibition and resistance process, and new analytical methods are an important means to improve resolution of this problem.